Liisa Nissinen

Collagenase-3 (MMP-13) is expressed by hypertrophic chondrocytes, periosteal cells, and osteoblasts during human fetal bone development.

Collagenase‐3 (MMP‐13) is a novel matrix metalloproteinase, the expression of which has so far only been documented in human breast carcinomas and osteoarthritic cartilage. In this study we have examined the expression of MMP‐13 during human fetal development. Northern blot hybridizations revealed abundant expression of MMP‐13 mRNAs in total RNA from fetal cartilage and calvaria at gestational age of 15 weeks. By in situ hybridization MMP‐13 transcripts were detected in chondrocytes of hypertrophic cartilage in vertebrae of the spinal column and in the dorsal end of ribs undergoing ossification, as well as in osteoblasts and periosteal cells below the inner periosteal region of ossified ribs. In contrast, no expression of MMP‐13 could be detected in osteoclasts. Furthermore, expression of MMP‐13 mRNA was detected in osteoblasts and fibroblasts primarily on the inner side of calvarial bone of the skull at 16 weeks of gestation. Expression of MMP‐13 mRNA by primary human fetal chondrocytes in culture was enhanced by transforming growth factor‐β (TGF‐β) and inhibited by bone morphogenetic protein‐2 (BMP‐2). No expression of MMP‐13 mRNA could be noted in other fetal tissues, including the skin, lungs, neural tissue, muscle, and liver. These results suggest that MMP‐13 plays an important role in the extracellular matrix remodeling during fetal bone development both via endochondral and intramembranous ossification. Dev. Dyn. 208:387–395, 1997.

Internalization of Echovirus 1 in Caveolae

ABSTRACT Echovirus 1 (EV1) is a human pathogen which belongs to the Picornaviridae family of RNA viruses. We have analyzed the early events of infection after EV1 binding to its receptor α2β1 integrin and elucidated the route by which EV1 gains access to the host cell. EV1 binding onto the cell surface and subsequent entry resulted in conformational changes of the viral capsid as demonstrated by sucrose gradient sedimentation analysis. After 15 min to 2 h postinfection (p.i.) EV1 capsid proteins were seen in vesicular structures that were negative for markers of the clathrin-dependent endocytic pathway. In contrast, immunofluorescence confocal microscopy showed that EV1, α2β1 integrin, and caveolin-1 were internalized together in vesicular structures to the perinuclear area. Electron microscopy showed the presence of EV1 particles inside caveolae. Furthermore, infective EV1 could be isolated with anti-caveolin-1 beads 15 min p.i., confirming a close association with caveolin-1. Finally, the expression of dominant negative caveolin in cells markedly inhibited EV1 infection, indicating the importance of caveolae for the viral replication cycle of EV1.

Integrin α2β1 promotes activation of protein phosphatase 2A and dephosphorylation of Akt and glycogen synthase kinase 3β

ABSTRACT Serine/threonine kinase Akt is a downstream effector protein of phosphatidylinositol-3-kinase (PI-3K). Many integrins can function as positive modulators of the PI-3K/Akt pathway. Integrin α2β1 is a collagen receptor that has been shown to induce specific signals distinct from those activated by other integrins. Here, we found that, in contrast what was found for cells adherent to fibronectin, α2β1-mediated cell adhesion to collagen leads to dephosphorylation of Akt and glycogen synthase kinase 3β (GSK3β) and concomitantly to the induction of protein serine/threonine phosphatase 2A (PP2A) activity. PP2A activation can be inhibited by mutation in the α2 cytoplasmic domain and by a function-blocking anti-α2 antibody. Akt can be coprecipitated with PP2A, and coexpression of Akt with PP2Ac (catalytic subunit) inhibits Akt kinase activity. Integrin α2β1-related activation of PP2A is dependent on Cdc42. These results indicate that cell adhesion to collagen modulates Akt activity via the α2β1-induced activation of PP2A.

The Fibril-associated Collagen IX Provides a Novel Mechanism for Cell Adhesion to Cartilaginous Matrix

Collagen IX is the prototype fibril-associated collagen with interruptions in triple helix. In human cartilage it covers collagen fibrils, but its putative cellular receptors have been unknown. The reverse transcription-PCR analysis of human fetal tissues suggested that based on their distribution all four collagen receptor integrins, namely α1β1, α2β1, α10β1, and α11β1, are possible receptors for collagen IX. Furthermore primary chondrocytes and chondrosarcoma cells express the four integrins simultaneously. Chondrosarcoma cells, as well as Chinese hamster ovary cells transfected to express α1β1, α2β1, or α10β1 integrin as their only collagen receptor, showed fast attachment and spreading on human recombinant collagen IX indicating that it is an effective cell adhesion protein. To further study the recognition of collagen IX we produced recombinant αI domains in Escherichia coli. For each of the four αI domains, collagen IX was among the best collagenous ligands, making collagen IX exceptional compared with all other collagen subtypes tested so far. Rotary shadowing electron microscopy images of both α1I- and α2I-collagen IX complexes unveiled only one binding site located in the COL3 domain close to the kink between it and the COL2 domain. The recognition of collagen IX by α2I was considered to represent a novel mechanism for two reasons. First, collagen IX has no GFOGER motif, and the identified binding region lacks any similar sequences. Second, the α2I domain mutations D219R and H258V, which both decreased binding to collagen I and GFOGER, had very different effects on its binding to collagen IX. D219R had no effect, and H258V prevented type IX binding. Thus, our results indicate that collagen IX has unique cell adhesion properties when compared with other collagens, and it provides a novel mechanism for cell adhesion to cartilaginous matrix.

Integrin and dystrophin associated adhesion protein complexes during regeneration of shearing-type muscle injury

In shearing injury both the myofibres and connective tissue framework are breached and the muscle tendon continuity is disrupted. During regeneration the firm myofibre to extracellular matrix (ECM) adhesion must be re-established. We have analysed the expression of selected molecules implementing this adhesion in regenerating myofibres 2-56 days after transection of rat soleus muscle using quantitative immunohistochemistry and Northern blotting. Beta1 integrin mRNA level and alpha7 integrin and vinculin immunoreactivities were transiently increased in both the intact and regenerating parts of the transected myofibres by day 5-7 with normalization by day 10-14. After day 14, alpha7 integrin and vinculin accumulated at the tips of the regenerating myofibres, indicating formation of new mini-myotendinous junctions (mMTJ). Immunoreactivities for dystrophin and associated proteins as well as merosin appeared in regenerating myotubes by day 3-4 reaching control levels by day 56. Our results suggest that integrin and dystrophin associated molecules are complementary in myofibre-ECM adhesion. During regeneration, ruptured myofibres temporarily reinforce their integrin mediated lateral adhesion until mMTJs are formed. Thereby the load on the newly formed scar and the risk of rerupture are reduced. Dystrophin associated molecules appear later and replace integrin on the lateral aspects, while both complexes are abundant at the mMTJs. These molecular events correspond to our previous results on tensile strength.

Molecular mechanism of α2β1 integrin interaction with human echovirus 1

Conformational activation increases the affinity of integrins to their ligands. On ligand binding, further changes in integrin conformation elicit cellular signalling. Unlike any of the natural ligands of α2β1 integrin, human echovirus 1 (EV1) seemed to bind more avidly a ‘closed’ than an activated ‘open’ form of the α2I domain. Furthermore, a mutation E336A in the α2 subunit, which inactivated α2β1 as a collagen receptor, enhanced α2β1 binding to EV1. Thus, EV1 seems to recognize an inactive integrin, and not even the virus binding could trigger the conformational activation of α2β1. This was supported by the fact that the integrin clustering by EV1 did not activate the p38 MAP kinase pathway, a signalling pathway that was shown to be dependent on E336‐related conformational changes in α2β1. Furthermore, the mutation E336A did neither prevent EV1 induced and α2β1 mediated protein kinase C activation nor EV1 internalization. Thus, in its entry strategy EV1 seems to rely on the activation of signalling pathways that are dependent on α2β1 clustering, but do not require the conformational regulation of the receptor.

Expression of α7β1 Integrin Splicing Variants during Skeletal Muscle Regeneration

Integrin α7β1 is a laminin receptor, both subunits of which have alternatively spliced, developmentally regulated variants. In skeletal muscle β1 has two major splice variants of the intracellular domain (β1A and β1D). α7×1 and α7×2 represent variants of the α7 ectodomain, whereas α7A and α7B are variants of the intracellular domain. Previously we showed that during early regeneration after transection injury of muscle α7 integrin mediates dynamic adhesion of myofibers along their lateral aspects to the extracellular matrix. Stable attachment of myofibers to the extracellular matrix occurs during the third week after injury, when new myotendinous junctions develop at the ends of the regenerating myofibers. Now we have analyzed the relative expression of β1A/β1D and α7A/α7B and α7×1/α7×2 isoforms during regeneration for 2 to 56 days after transection of rat soleus muscle using reverse transcriptase-polymerase chain reaction and immunohistochemistry. During early regeneration β1A was the predominant isoform in both the muscle and scar tissue. Expression of muscle-specific β1D was detected in regenerating myofibers from day 4 onwards, ie, when myogenic mitotic activity began to decrease, and it became more abundant with the progression of regeneration. α7B isoform predominated on day 2. Thereafter, the relative expression of α7A transcripts increased until day 7 with the concomitant appearance of α7A immunoreactivity on regenerating myofibers. Finally, α7B again became the predominant variant in highly regenerated myofibers. Similarly as in the controls, α7×1 and α7×2 isoforms were both expressed throughout the regeneration with a peak in α7×1 expression on day 4 coinciding with the dynamic adhesion stage. The results suggest that during regeneration of skeletal muscle the splicing of β1 and α7 integrin subunits is regulated according to functional requirements. α7A and α7×1 appear to have a specific role during the dynamic phase of adhesion, whereas α7B, α7×2, and β1D predominate during stable adhesion.

A small-molecule inhibitor of integrin α2β1 introduces a new strategy for antithrombotic therapy

Interaction of blood platelets with vascular collagen is an initiating event in haemostasis and thrombus formation. Based on molecular modelling of human integrin alpha2I domain and cell-based screening assays we have developed sulfonamide derivatives, a mechanistically novel class of molecules. These molecules show antiplatelet efficacy by selectively inhibiting alpha2beta1 integrin-mediated collagen binding. One sulfonamide derivative, named BTT-3016, showed inhibitory capacity in several assessments of human platelet interaction with collagen. It inhibited about 90% of the aggregation of gel-filtered magnesium-supplemented platelets and 70% of aggregation in PPACK-anticoagulated platelet-rich plasma when stimulated with collagen but not with ADP. The antiplatelet activity of BTT-3016 was dependent on alpha2beta1 integrin, since in collagen binding test BTT-3016 had no effect on the platelets derived from alpha2 integrin null mice. When tested in an in vivo model in mice, BTT-3016 clearly reduced thrombus formation on the vessel wall after vascular injury. Furthermore, BTT-3016 prolonged tail-bleeding time in a manner comparable to aspirin. We show that new alpha2beta1 inhibitors exert collagen-specific antiplatelet activity and regulate thrombus growth in vivo without compromising primary haemostasis more than aspirin. We suggest that the alpha2beta1 inhibiting strategy could be further developed for the prevention and treatment of arterial thrombosis.

Calpain 1 and 2 Are Required for RNA Replication of Echovirus 1

ABSTRACT Calpains are calcium-dependent cysteine proteases that degrade cytoskeletal and cytoplasmic proteins. We have studied the role of calpains in the life cycle of human echovirus 1 (EV1). The calpain inhibitors, including calpeptin, calpain inhibitor 1, and calpain inhibitor 2 as well as calpain 1 and calpain 2 short interfering RNAs, completely blocked EV1 infection in the host cells. The effect of the inhibitors was not specific for EV1, because they also inhibited infection by other picornaviruses, namely, human parechovirus 1 and coxsackievirus B3. The importance of the calpains in EV1 infection also was supported by the fact that EV1 increased calpain activity 3 h postinfection. Confocal microscopy and immunoelectron microscopy showed that the EV1/caveolin-1-positive vesicles also contain calpain 1 and 2. Our results indicate that calpains are not required for virus entry but that they are important at a later stage of infection. Calpain inhibitors blocked the production of EV1 particles after microinjection of EV1 RNA into the cells, and they effectively inhibited the synthesis of viral RNA in the host cells. Thus, both calpain 1 and calpain 2 are essential for the replication of EV1 RNA.

Complement Factor I Promotes Progression of Cutaneous Squamous Cell Carcinoma

The incidence of cutaneous squamous cell carcinoma (cSCC) is rising worldwide. We have examined the role of complement components in the progression of cSCC. Analysis of cSCC cell lines (n=8) and normal human epidermal keratinocytes (n=11) with whole transcriptome profiling (SOLiD), quantitative real-time reverse transcriptase-PCR, and western blotting revealed marked overexpression of complement factor I (CFI) in cSCC cells. Immunohistochemical analysis for CFI in vivo showed stronger tumor cell-specific labeling intensity in invasive sporadic cSCCs (n=83) and recessive dystrophic epidermolysis bullosa-associated cSCCs (n=7) than in cSCC in situ (n=65), premalignant epidermal lesions (actinic keratoses, n=64), benign epidermal papillomas (seborrheic keratoses, n=39), and normal skin (n=9). The expression of CFI was higher in the aggressive Ha-ras-transformed cell line (RT3) than in less tumorigenic HaCaT cell lines (HaCaT, A5, and II-4). The expression of CFI by cSCC cells was upregulated by IFN-γ and IL-1β. Knockdown of CFI expression inhibited proliferation and migration of cSCC cells and resulted in inhibition of basal extracellular signal-regulated kinase (ERK) 1/2 activation. Knockdown of CFI expression potently inhibited growth of human cSCC xenograft tumors in vivo. These results provide evidence for the role of CFI in the progression of cSCC and identify it as a potential therapeutic target in this nonmelanoma skin cancer.